Tobacco dependence and chronic pain are two highly prevalent and comorbid conditions that engender substantial burdens upon individuals and systems. Pain has been shown to motivate smoking, pain patients endorse smoking for pain-coping, and recurring pain may serve as a significant barrier to smoking cessation. Although animal studies have consistently demonstrated increased pain in the context of nicotine deprivation, we are not aware of any studies that tested whether smoking abstinence or nicotine withdrawal may increase pain reactivity in humans. Given the clinical implications of experiencing increased pain as a consequence of abstaining from tobacco, human trials are clearly warranted. Therefore, the main goal of the proposed study is to test, experimentally, the effects of smoking abstinence on self-reported and physiological pain responding among nicotine-dependent tobacco smokers. Participants (N = 198) will be randomized to one of three experimental conditions prior to undergoing pain induction: (1) 24-hour smoking abstinence (SA); (2) two-hour minimal deprivation (MD); and (3) continued smoking (CS). Specifically, we hypothesize that smokers randomized to the SA condition will report lower pain threshold, greater pain intensity, and greater pain unpleasantness during experimental pain induction than smokers randomized to the MD condition, who will, in turn, report lower pain threshold, greater pain intensity, and greater pain-related unpleasantness than smokers randomized to the CS condition (i.e., SA > MD > CS). We also hypothesize that symptoms characteristic of nicotine withdrawal will mediate the relationship between smoking abstinence and pain responding, such that withdrawal severity will be positively associated with increased pain responding. A secondary aim of the proposed study is to explore the influence of additional mechanisms that may mediate or moderate the effect of smoking abstinence on pain responding. We conceptualize the currently proposed study as a prototypical example of translational, cross-disciplinary research that has the potential to inform the development of targeted interventions. We also believe the proposed project will yield findings that enhance scientific knowledge within the medical and behavioral sciences, inform clinical practice with regard to the treatment of both pain and smoking, and challenge current clinical practice paradigms. For example, given evidence that pain is a motivator of smoking, smoking cessation interventions for persons with comorbid pain disorders may be modified to account for the antithetical influence of smoking abstinence-induced amplification of pain. Finally, the current lack of a human laboratory model of pain and smoking that allows for manipulation of both smoking and pain represents a critical barrier to progress in this broad domain.